This invention relates to compositions and methods for producing facile perfusion of active agents in tissues following direct intra-tissue delivery. In another aspect it relates to compositions and methods to identify those compositions capable of producing novel pharmacological activities of an active agent following intra-tissue delivery.
The inability of agents to perfuse throughout a tissue, permeate cells and achieve biologically effective levels at their targets has been a longstanding problem in the field of drug delivery. Methods to overcome this include targeting of active agents to tissues by:
Use of delivery vehicles which are targeted to specific receptors on the surface of cells (e.g. monoclonal antibodies,)xe2x80x94this is limited by the inadequate diffusion of the antibody-active agent conjugates through solid tissue.
Use of active agents whose targets are only expressed in target cells (e.g. DNA with tissue specific promoters)xe2x80x94these are limited by the inability of vectors delivering these agents to access large numbers of cells through solid tissue, production of efficacious amounts of active agents and potential dangers which viral vectors/DNA presents in the target and surrounding cells.
Conjugating the active agent to low density lipoprotein (LDL) for internalization via the cell membrane LDL receptor has been attempted to overcome physiological barriers which obstruct systemic drug delivery to target tissue (e.g. capture of agents by proteins in solution in the blood and the inability of many agents to effectively cross the blood-brain barrier). Again, this results in a lack of specificity of agent delivery and problems in obtaining adequate amounts of agents at their target sites. High flow microinfusion has been attempted to achieve an increase in drug delivery across the blood-brain barrier.
Use of liposomes to encapsulate agents in the hydrophilic aqueous interspaces or in the lipid bilayer for hydrophobic compounds is another delivery method limited by the inability to achieve adequate targeting and perfusion through solid tissue.
Antineoplastic solutions consisting of an organic water miscible solvent vehicle containing an antineoplastic agent solute have been previously shown to be effective for the rapid and effective perfusion of solid tumors (Pietronigro 1991 U.S. Pat. No. 5,051,257 and Pietronigro 1992, U.S. Pat. No. 5,162,115).
The effective perfusion through non-neoplastic tissues, however, remains a problem. Non-neoplastic mammalian tissues are tissues or tissue regions not comprised entirely of a new and abnormal formation of tissue such as a solid palpable tumor, but rather, a tissue comprised essentially of original normal tissue which now, in part, contains: abnormal (i.e., damaged or diseased) tissue components, e.g., dispersed, individual cancerous cells, ischemic cells, thrombosed blood vessels, cells expressing specific aberrant gene products(s), cells producing specific gene products in abnormal quantities or unnatural extracellular accumulations of substances; and/or foreign elements, e.g., infectious agents such as bacteria and viruses and fungi. Mixed tissues are tissues containing both regions of normal tissue and regions of non-neoplastic tissues.
While the structure of solid tumors is ideal for penetration by an organic water miscible solvent vehicle, delivering the agent to cells throughout the tumor, the structure of normal, non-neoplastic and mixed tissues presents many obstacles to effective penetration by active agents.
Tumors possess large quantities of interstitial space which can be up to six times greater than that found in normal tissue (Jain 1987, Cancer Research, 47, 3039-3051). A contributing factor to the increased interstitial space in tumors is the lower density of cells present. Cellular architecture and organization also varies greatly between tumors and normal tissue. The structural integrity found in normal tissues is a result of the presence of cell-cell junctions (e.g., tight junctions, desmosomes), cell-cell surface adhesion molecules (e.g., cadherins) and cell-basement membrane adhesion molecules (e.g. integrins). The cell-cell and cell-basement membrane attachments are reduced in tumors as a result of a decrease in the expression of molecules responsible for these interactions (Dvorak 1998, Critical issues in Tumor Microcirculation, Angiogenesis and Metastasis, Lecture 1, 1-17, St. Croix 1997, Current Opinion in Oncology, 9, 549-556). The cells in tumors grow in small clumps distributed throughout the interstitial space, with little cell-cell attachment, or structural integrity. Normal tissue consists of highly organized three-dimensional arrangements of cells, often many cell layers thick.
Tumor blood vessels lack the structural integrity of blood vessels in normal tissue. Tumor vessels are hyperpermeable (Dvorak 1998, Critical Issues in Tumor Microcirculation, Angiogenesis and Metastasis, Lecture 1, 1-17), and lack the normal basement membrane surrounding the vessels in normal tissue. The lymphatic system present in normal tissue is lacking in tumors. Fluid in the interstitial space is drained by lymphatic vessels in normal tissue. In tumors, there is a build up of fluid in the interstitium due to the absence of lymphatics and the hyperpermeability of the blood vessels which results in an increase in the pressure in the interstitial space. A pressure gradient exists in tumors, the highest pressure being at the tumor center and which decreases towards the edge of the tumor (Jain, 1997, Microcirculation, 4(1), 1-23).
The low cell density and increased interstitial space of solid tumors provides an ideal medium for the organic water miscible solvent vehicle to penetrate through. Arrangement of cells in clumps with little adhesion between them enables all cells throughout the tumor to be perfused with the antineoplastic solution. The interstitial pressure gradient that exists from the center to the outer edge of the tumor would aid perfusion of an antineoplastic solution injected into the tumor center.
In contrast non-neoplastic tissues having relatively small interstitial space coupled with the rigid cellular organization and structural integrity present many barriers to the transport of an active agent administered by direct injection. The high degree of cell-cell and cell-basement membrane interactions, high cell density and many cell layers present physical barriers to the distribution of an active agent. The lack of a pressure gradient is also believed to act to lessen distribution of a solution throughout the tissue. Thus, the effective perfusion through non-neoplastic tissues, normal tissue or mixed tissues remains a problem.
In view of the above, the limitations to the perfusion of tissues known to the prior art are readily apparent.
Accordingly it is an object of the present invention to provide compositions and a method for the facile perfusion of an active agent throughout a tissue.
Another object of the present invention is to provide compositions and a method to selectively affect a subset of tissue components by the facile perfusion of an active agent throughout a tissue.
Another object of the present invention is to provide compositions and a method to treat a tissue with a high (previously unobtainable) dose of an active agent.
Another object of the present invention is to provide compositions and a method for producing novel pharmacological activities of active agents in tissues.
Another object of the present invention is to provide compositions and a method to alter the spectrum of activity of an active agent attainable in vivo.
Another object of the present invention is to provide a method for the identification of novel pharmacological activities of an active agent.
Other objects and advantages of the present invention will, in part, be apparent and will, in part, appear hereinafter.
The manner in which these and other objects are realized by the present invention will be apparent from the summary and detailed description set forth below.
In accordance with the present invention facile perfusion of a mammalian tissue with an active agent is achieved by direct intra-tissue injection of solutions consisting of active agent solutes in water miscible organic solvent vehicles. The active agents are chosen to have solubility, stability and bioavailability in the water miscible organic solvent vehicle. Intra-tissue injection of these solutions permits high levels of active agent solute to readily permeate through the tissue and into cells thereby achieving heretofore unobtainable levels at target sites. The facile perfusion results in high degrees of efficacy and can produce selective effects on the abnormal tissue components and foreign elements of the tissue perfused and can also produce novel pharmacological or biochemical actions previously unknown or unattainable in vivo due to the high levels of active agent delivered to the tissue.